Phenolic amino-carboxylic acid complexes for forming radiopharmaceuticals

ABSTRACT

A complex of a chelating agent is formed and labelled with technetium-99m. The chelating agent is either an ethylenediamine carboxylic acid or its salt having a phenolic group at each end of the molecule such as EDDHA and HBED or alternatively the phenolic amino carboxylic acid or its salt having a single phenolate group such as HBG or HBS. The complex is a stannous complex which when labelled with technetium-99m has a good balance of lipo-solubility and waater-solubility to permit hepato-biliary imaging. The radiopharmaceutical may be useful for assessing hepatobiliary function in animals including human beings. A particularly useful chelating agent is ethylene-bis-(α-imino-2-hydroxy-5-bromophenyl acetic acid).

REFERENCE TO CO-PENDING APPLICATION

This application is a Continuation-in-part based on U.S. PatentApplication Ser. No. 655 034 filed Feb. 3, 1976.

The present invention relates to radiopharmaceuticals and complexeswhich can be labelled to produce radioactive complexes. The radioactivecomplexes are to be used to assess the function of certain animal organsby scintigraphic means.

More specifically, in one aspect the invention is concerned with metalchelates in which the metal is a pharmaceutically acceptable radioactiveisotope, which is selected and designed so as to be distributed in acertain way when administered to an animal, whereby imaging of certainselected organs and assessment of their function can take place.

Radiopharmaceuticals have previously been used as imaging agents forinvestigating the function of organs in animals including human beings.Complexes of radioactive isotopes have been used by administering thecomplex, usually intravenously, with the result that the material iscarried by the bloodstream to locate preferentially but temporarily insome particular organ. By the use of suitable imaging equipment, thepassage of the compound through the organ can be observed and deductionscan be made as to whether the organ is functioning satisfactorily or hasany defects. After the administration of a dose, the passage of thematerial is imaged by using known scintigraphic apparatus.

Certain body functions can be imaged by the use of a radioactive complexformed from the known chelating agent ethylenediaminetetracetic acid(EDTA). EDTA is water-soluble and U.S. Pat. No. 3,466,361 describes theuse of a technetium complex of EDTA for kidney imaging. However, thiscomplex is not usable where lipo-solubility is necessary for imagingcertain organs.

The present invention is concerned with the problem of providing newradioactive imaging agents which are lipo-soluble and which could beadministered to animals including human beings. There is need for newimaging agents to be provided which have low toxicity, a reduction inthe type and level of any side effects, convenience of use, greatereconomy, rapid preferential location in animal and a high degree ofpreferential location.

The present invention is particularly directed to the provision of novelradiopharmaceuticals for assessing hepato-billary function.

The present invention provides in one aspect such a radiopharmaceuticalas a metal chelate in which the metal is a pharmaceutically acceptableradio isotope for an animal and a chelating agent is a phenolicaminocarboxylic acid or an alkali metal salt thereof.

Embodiments of the invention include material derived from a chelatingagent having a single phenolate group although as will be explainedhereinafter it appears that better results are derived from the use of achelating agent which is an ethylenediamine carboxylic acid or its salt,which has a phenolic group at each end of the molecule.

A particularly important group of chelating agents for use inembodiments of the present invention is the group consisting of or basedon the following compounds: ##STR1## where X is hydrogen, a halogen oran alkyl group, this compound being known as ethylene-bis(α-imino-o-hydroxyphenylacetic acid) referred to as EDDHA: ##STR2##wherein X is hydrogen, halogen or an alkyl group, this compound beingknown as N,N-di(2-hydroxybenzyl) ethylene-diamine-N,N-diacetic acid andreferred to hereinafter as HBED:

(3) hydroxy benzyl glycine hereinafter referred to as HBG and having theformula: ##STR3## (4) Hydroxyl benzyl sarcosine, hereinafter referred toas HBS and having the formula: ##STR4##

The invention is preferably used with the metal being technetium-99mwhich is known to be an acceptable radio isotope for pharmaceuticaluses. Indium 113m has also been successfully tested, but technetium ispreferred.

The present invention is primarily concerned with the provision of animaging agent for investigation of hepato-biliary function in animalsincluding human beings. According to the invention, a suitable metalradio isotope is chosen and is applied to a chelating agent selected forits capacity to form an effective metal chelate which has a good balanceof aqueous and lipid solubility and will preferentially locate bothrapidly and with a high degree of preference in the liver of an animalfor excretion along the common bile duct whereby the passage of thematerial with time can be monitored by imaging with conventionalscintigraphic apparatus for the purpose of determining the function ofthe liver and bile duct. It is known that the liver has the capacity toremove fat soluble components and thus liposolubility is required, butin order to best administer a metal chelate embodying the invention, itshould be water soluble so as to permit injection into the blood streamwhereby rapid results can be obtained.

In preferred embodiments of the invention, the metal chelate is formedfrom a substituted derivative of the chelating agent. Advantageously thederivative is ethylene-bis-(α-imino-o-hydroxyphenylacetic acid) (EDDHA).

One particular derivative to be used in an important embodiment of theinvention is ethylene-bis-(α-imino-2-hydroxy-5-bromophenyl acetic acid),although other halogen substituted derivatives are also useful.

The invention in another aspect provides a novel stannous complex of achelating agent which is adapted to be labelled, for example bytechnetium 99m, prior to application to the patient. The stannouscomplex is either a complex of a phenolic aminocarboxylic acid chelatingagent containing an ethylenediamine dicarboxylic acid bridge and aterminal phenolic group at each end of said bridge or of a phenolicaminocarboxylic acid containing a single phenolate group and being bothlipo-soluble and water soluble.

It has been found that embodiments of the invention can be particularlyuseful for use as an imaging agent for observing the morphology andfunction of organs such as the liver and gall bladder in animals. It isthought that at least some embodiments of the invention may beparticularly useful in humans.

There is a great need for imaging agents which will permit organs suchas the gall bladder to be visualised.

It is considered that embodiments of the invention may be particularlyadvantageous in permitting a convenient imaging agent to be provided,the agent having an acceptably low toxicity and an advantageously highlevel of preferential location at an advantageous rate therebypermitting rapid and efficient diagnosis to be carried out with highlevels of safety.

Particularly when the metal chelate is formed from technetium-99m withthe chelating agent being EDDHA or HBED, it is thought that asurprisingly high degree of preferential location at a rapid rate isachieved thereby permitting rapid imaging to occur with low toxicity andlow side effects in the material, the rapid and highly preferentiallocation facilitating very small radioactive doses to be included in thematerial.

The manner in which technetium chelate embodying the invention is formedis not fully understood, but it is thought that each molecule ofchelating agent has a technetium atom which is bonded to the chelatingagent through a plurality of bonds. The nature of the bonding is notunderstood but it is thought that in the case of EDDHA and HBED thebonding would involve any two or all of the following: the two nitrogenatoms, the two hydroxy groups and the two carboxylic acid groups.

It is thought that the chelating agent has a high lipid solubility as aresult of the substituted phenolic ring structures in the molecule andit is this lipid solubility which results in the liver functioning toexcrete the complex. Thus, technetium is temporarily preferentiallylocated in the liver before passing to the gall bladder and duodenum.

Another aspect of the invention consists in a method of producing ametal chelate as described in any one of the forms mentioned above,characterised by adding a quantity of radionuclide to a tin (II) complexof the chelating agent, controlling the pH of the mixture and holdingthe mixture for a length of time sufficient to produce the desiredcomplex.

The present invention is useful in a method of treating animals in whichthe examination step comprises administering a pharmaceutical complexaccording to the first aspect of the invention or made according to thesecond aspect of the invention and observing the resultant manner inwhich the complex is distributed in the animal by the use ofscintigraphic apparatus.

One of the preferred chelating agents to be used according to thepresent invention is EDDHA compound the preparation of which has beenreported in U.S. Pat. No. 2,824,128.

A large number of EDDHA compounds can be made which can be useful in thepresent invention and the following list is given by way of example:

ethylene-bis-(α-imino-o-hydroxynaphthyl acetic acid)

ethylene-bis-(α-imino-2-hydroxy-3-chlorophenyl acetic acid)

ethylene-bis-(α-imino-2-hydroxy-5-chlorophenyl acetic acid)

ethylene-bis-(α-imino-2-hydroxy-5-iodophenyl acetic acid)

ethylene-bis-(α-imino-2-hydroxy-5-bromophenyl acetic acid)

ethylene-bis-(α-imino-2-hydroxy-3,5-dibromophenyl acetic acid)

ethylene-bis-(α-imino-2-hydroxy-3,5-dichlorophenyl acetic acid)

ethylene-bis-(α-imino-2-hydroxy-5-methylphenyl acetic acid)

ethylene-bis-(α-imino-2-hydroxy-3,5-dimethylphenyl acetic acid)

ethylene-bis-(α-imino-2-hydroxy-5-tertiarybutylphenyl acetic acid)

ethylene-bis-(α-imino-o-hydroxyphenyl acetic acid)

For the purpose of illustration examples will now be given of theproduction of metal chelates embodying the invention.

EXAMPLE 1

1 ml of a normal saline or aqueous solution containing technetium-99m inthe form of pertechnetate was added to a preformed complex made by theaddition of 250 μg to 1 mg of SnCl₂.2H₂ O to 15mg ofethylene-bis-(α-imino-2-hydroxy-5-chlorophenyl acetic acid) in 5 ml ofH₂ O at pH 8-9. The solution was stirred for 5-10 minutes, and finallythe product was filtered through a membrane to sterilise the product.

EXAMPLE 2

1 to 2 ml of sodium pertechnetate was added to a preformed, sterile,freeze-dried complex containing 3 mg ofethylene-bis-(α-imino-2-hydroxy-5-chlorophenyl acetic acid) and 50-200μg of SnCl₂.2H₂ O. The resultant solution is ready for injection.

Other examples of the invention may be based on chelating agents of theHBED type, examples of which are as follows:

N,n'-bis-(2-hydroxy-3-chlorobenzyl)ethylenediamine diacetic acid

N,n'-bis-(2-hydroxy-5-chlorobenzyl)ethylenediamine diacetic acid

N,n'-bis-(2-hydroxy-3,5-dibromobenzyl)ethylenediamine diacetic acid

N,n'-bis-(2-hydroxy-3,5-dichlorobenzyl)ethylenediamine diacetic acid

N,n'-bis-(2-hydroxy-5-iodobenzyl)ethylenediamine diacetic acid

N,n'-bis-(2-hydroxy-5-bromobenzyl) ethylenediamine diacetic acid

N,n'-bis-(2-hydroxy-5-methylbenzyl) ethylenediamine diacetic acid

N,n'-bis-(2-hydroxy-3,5-dimethylbenzyl) ethylenediamine diacetic acid

N,n'-bis-(2hydroxy-5-tertiarybutylbenzyl) ethylenediamine diacetic acid

N,n'-bis-(2-hydroxy-benzyl) ethylenediamine diacetic acid

N,n'-bis-(2-hydroxy-naphthyl) ethylenediamine diacetic acid

Specific examples will now be given for further illustration.

EXAMPLE 3

1 ml of a normal saline or aqueous solution containing technetium-99m inthe form of pertechnetate was added to a preformed complex made by theaddition of 250μg to 1mg of SnCl₂ 2H₂ O to 15 mg ofN,N'-bis-(2-hydroxy-5-bromo benzyl) ethylenediamine diacetic acid in 5ml of H₂ O at pH 8-9. The solution was stirred for 5-10 minutes, andfinally the product was filtered through a membrane to sterilise theproduct.

EXAMPLE 4

1 to 2 ml of sodium pertechnetate was added to a preformed sterile,freeze-dried complex containing 3mg ofN,N'-bis-(2-hydroxy-5-bromobenzyl) ethylenediamine diacetic acid and50-200 μg of SnCl₂ 2H₂ O, The resultant solution is ready for injectionTests have been conducted on animals; the product of each of theexamples given above was administered in a pharmaceutical dose anduseful images were obtained showing initially the preferentiallocalisation of the complex in the liver and subsequent advance of thecomplex to the gall bladder and with the passage of further time theimages showed passage of the material through the duodenum.

The formation of EDDHA, HBED, HBG and HBS are known and described in thefollowing publications:

EDDHA: U.S. Pat. No. 2824128 - Inventors M. Dexter and R.J. Cranston.

HBED: British Patent 843000 - Inventor - Geigy A.G.

HBG and HBS: Czechoslovakian Patent No. 94067 - Inventors- J. Korbl andV. Svoboda.

EXAMPLE 5

3 grams of the appropriate chelating agent (EDDHA, HBED, HBS, HBG orsubstituted derivative thereof) were dissolved in 20 ml of 1M sodiumhydroxide and the solution diluted to 250 ml with water for injection. Anitrogen purge of the solution was commenced and 100 mg of stannouschloride dihydrate added directly to the solution. When the stannouschloride had completely dissolved, the pH of the solution was adjustedto 8.75 ± 0.2 using 1M hydrochloric acid.

The solution was then diluted to 1000 ml with water for injection andfiltered through 0.22 μM membrane filter. One millilitre aliquots of thesolution were then dispensed into 2 ml vials whilst continuing thenitrogen purge throughout the dispensing. Sterile lyophilising stopperswere then inserted part way into the vials before transfer tofreeze-drying equipment. Freeze-drying was commenced, the final productafter 36 hours consisting of a sterile lyophilised powder sealed undervacuum.

We claim:
 1. A reagent which is adapted to be labelled withtechnetium-99m for use as a hepato-biliary imaging radiopharmaceuticalfor use on an animal, the reagent comprising a stannous complex of aphenolic aminocarboxylic acid chelating agent selected from the groupconsisting of an ethylene diamine di(hydroxy phenyl carboxylic acid) anda di (hydroxy benzyl) ethylene diamine dicarboxylic acid.
 2. A reagentas claimed in claim 1, wherein said chelating agent is selected from thegroup consisting of ##STR5## wherein X is hydrogen, a halogen or analkyl group and ##STR6## wherein X is hydrogen, a halogen or an alkylgroup.
 3. A reagent which is adapted to be labelled with technetium-99mfor use as a hepato-biliary imaging radiopharmaceutical for use on ananimal, the reagent comprising a stannous complex of a phenolicaminocarboxylic acid chelating agent containing a single phenolic group,having a balance of both lipo-soluble and water-soluble properties.
 4. Areagent as claimed in claim 3, wherein said chelating agent is selectedfrom the group consisting of hydroxybenzyl glycine and hydroxybenzylsarcosine.
 5. A reagent as claimed in claim 4, wherein said phenolicaminocarboxylic acid has lipo-solubilizing substituents in the phenolicring.
 6. A reagent as claimed in claim 5, wherein said lipo-solubilisingsubstituent is a halogen.
 7. A reagent as claimed in claim 2, whereinthe reagent is a stannous chelate ofethylene-bis-(α-imino-2-hydroxy-5-bromophenyl acetic acid).
 8. A reagentas claimed in claim 2, wherein the reagent is a stannous chelate ofN,N'-bis-(2-hydroxy-5-bromobenzyl ethylenediamine diacetic acid).
 9. Areagent which is adapted to be labelled with technetium-99m for use as ahepato-biliary imaging radiopharmaceutical on an animal, the reagentbeing a stannous chelate of a chelating agent selected from the groupconsisting of hydroxybenzyl glycine and hydroxybenzyl sarcosine havinglipo-solubilizing substituents in the phenolic ring and having aliphaticsubstituents.
 10. A reagent as claimed in claim 9, wherein saidlipo-solubilising substituents are selected from the halogens.
 11. Areagent as claimed in claim 4, wherein the reagent is a stannous chelateof N-(o-hydroxybenzyl)glycine.
 12. A reagent as claimed in claim 4,wherein the reagent is a stannous chelate ofN-(o-hydroxybenzyl)sarcosine.
 13. A method of formingradiopharmaceuticals for assessing hepato-biliary function in animals,comprising reacting a stannous chelate of a chelating agent selectedfrom the group consisting of lipo-solubilizing ring substitutedderivatives of ##STR7## wherein X is hydrogen, a halogen or an alkylgroup, ##STR8## wherein X is hydrogen, a halogen or an alkyl group, (3)hydroxybenzyl glycine and(4) hydroxybenzyl sarcosinewith a radio isotopeselected from the group consisting of ¹¹¹ In or ^(113m) In and ^(99m)Tc.
 14. A radiopharmaceutical for assessing hepato-biliary functioncomprising a lipo-soluble metal chelate in which the metal is apharmaceutically acceptable radio isotope for an animal and the metalchelate is formed from ethylene-bis(α-imino-2-hydroxy-5-bromophenylacetic acid).
 15. A radiopharmaceutical for assessing hepato-biliaryfunction comprising a lipo-soluble metal chelate in which the metal is apharmaceutically acceptable radio isotope for an animal and the metalchelate is formed from a stannous complex of a chelating agent selectedfrom the group consisting of ##STR9## wherein X is hydrogen, a halogenor an alkyl group and ##STR10## wherein X is hydrogen, a halogen or analkyl group.